This proposal brings together investigators from Rutgers University (Tumer, Kimball, Augery), Albert Einstein College of Medicine (Schramm, Almo and Cameron) and Wadsworth Center (Mantis) with expertise in biochemistry, structural biology, medicinal chemistry, and toxicology to identify inhibitors that target ribosome interactions and catalytic activity of ricin. Currently, there is no proven, safe treatment for ricin intoxication or infection by related Shiga toxin producing Shigella or E. coli. Two ricin vaccines in clinical trials do not elicit robust toxin neutralizing activity. The goal of this proposal is to fill this gap by by identifying peptides and small molecule fragments that bind to key pockets on ricin A chain (RTA) and inhibit its activity. During the previous funding period we identified the host target of ricin as the conserved C-terminal 11-mer (P11) of the ribosomal P-protein stalk. We showed that the ribosome binding surface of RTA is on the opposite face of the active site cleft and proposed a model where binding to the ribosomal stalk stimulates ribosome depurination by reorienting the active site of RTA towards the SRL. These studies established a new paradigm for the mechanism of depurination and identified toxin/ribosome interactions as a new target for inhibitor discovery. We recently discovered a new hydrophobic pocket anchored by an essential arginine critical for ribosome interactions of RTA. Our overall hypothesis is that we can inhibit the catalytic activity and the toxicity of ricin by interfering with ribosome interactions of RTA. We will identify the key interacting residues at the ribosome binding surface of RTA as a starting point in inhibitor discovery. Using peptide arrays of the ribosomal target we will identify peptide analogs that bind to the ribosome binding surface and block the ribosome interactions of RTA. We will use fragment based lead discovery (FBLD) with surface plasmon resonance (SPR) to identify fragments that can bind to the ribosome binding pocket, the active site cleft or previously unidentified pockets and inhibit the depurination activity and toxicity of RTA. Ribosome binding and active site mutants will be used to determine the binding site selectivity and X-ray crystal structure analysis will be used to elucidate the binding mode of the peptides and the fragments. The fragment hits will be optimized and evaluated in cell-based assays and in a mouse model of ricin intoxication. The promising leads will be used as a starting point to build a scaffold. Our innovative approach, rigorous methodology and deep insight into the structure function analysis of RTA will provide new knowledge about the basic mechanism for molecular recognition of the stalk and will help identify inhibitors that can be used as new leads for future therapeutic design.